Human cervical cancer protooncogene and protein encoded therein

ABSTRACT

A human cervical cancer 1 protooncogene having a base sequence of SEQ ID:1 or a fragment thereof is overexpressed in various cancer tissues and can be used in diagnosing various cancers and an anti-sense gene complementary thereto can be used in treating cancers.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation patent application of PCT PatentApplication No. PCT/KR00/00284, which was filed on Mar. 30, 2000,designating the United States of America.

FIELD OF THE INVENTION

The present invention relates to a novel protooncogene and proteinencoded therein, and more particularly, to a human cervical cancer 1protooncogene and a protein derived therefrom, which can be used indiagnosis of various cancers.

BACKGROUND OF THE INVENTION

Higher animals including man each carry approximately 100,000 genes, butonly about 15% thereof is expressed, and characteristics of individual'sbiological process, e.g., genesis, differentiation, homeostasis,responses to stimuli, control of cell segmentation, aging and apoptosis(programmed cell death), are determined depending on which genes areexpressed (see Liang, P. and A. B. Pardee, Science, 257: 967-971(1992)).

Pathogenic phenomena such as tumorigenesis are caused by gene mutationwhich brings about changes in the mode of gene expression. Therefore,comparative studies of gene expressions in various cells have beenconducted to provide bases for establishing viable approaches to theunderstanding of diverse biological phenomena.

For example, the mRNA differential display (DD) method suggested byLiang and Pardee is effective in elucidating the nature of tumorsuppressor genes, cell cycle-related genes and transcriptionalregulatory genes that control apoptosis (see Liang, P. and A. B. Pardeesupra). Further, the DD method has been widely used in examining theinterrelationship of various genes in a cell.

It has been reported that tumorigenesis is caused by various geneticchanges such as the loss of chromosomal heterozygosity, activation ofoncogenes and inactivation of tumor suppressor genes, e.g., p53 gene(see Bishop, J. M., Cell, 64: 235-248 (1991); and Hunter, T., Cell, 64:249-270 (1991)). Further, it has been reported that 10 to 30% of humancancer arises from the activation of oncogene through amplification ofprotooncogenes.

Therefore, the activation of protooncogenes plays an important role inthe etiology of many tumors and there has existed a need to identifyprotooncogenes.

The present inventor has endeavored to unravel examine the mechanisminvolved in the tumorigenesis of cervical cancer; and, has unexpectedlyfound that a novel protooncogene, human cervical cancer 1 (HCCR-1), isspecifically overexpressed in cancer cells. This protooncogene can beeffectively used in diagnosis, prevention and treatment of variouscancers, e.g., leukemia, lymphoma, kidney, liver, lung, ovary anduterine cervix cancers.

SUMMARY OF THE INVENTION

Accordingly, the primary object of the present invention is to provide anovel protoncogene and a fragment thereof.

Other objects of the present invention are to provide:

a recombinant vector containing said protooncogene or a fragment thereofand a microorganism transformed therewith;

a protein encoded in said protooncogene and a fragment thereof;

a kit for diagnosis of cancer containing said protooncogene or afragment thereof;

a kit for diagnosis of cancer containing said protein or a fragmentthereof;

an anti-sense gene having a base sequence complementary to that of saidprotooncogene or a fragment thereof; and

a process for treating or preventing cancer by using said anti-sensegene.

In accordance with one aspect of the present invention, there isprovided a novel protooncogene having the nucleotide sequence of SEQ IDNo:1 or a fragment thereof.

In accordance with another aspect of the present invention, there isprovided a recombinant vector containing said protooncogene or afragment thereof and a microorganism transformed with said vector.

In accordance with still another aspect of the present invention, thereis provided a protein having the amino acid sequence of SEQ ID No:2 or afragment thereof derived from said protooncogene or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings which respectivelyshow;

FIG. 1: the DD identification of altered gene expression in normalcervix tissue, primary cervical cancer tissue, metastatic lymph nodetissue and CUMC-6 cervical cancer cells.

FIG. 2: the prediction of the hydrophobicity of transmembrane regions inthe protooncogene of the present invention using TMPRED program.

FIG. 3: the results of northern blot analyses for HCCR-1 gene expressedin normal cervical tissues, cervical cancer tissues and cervical cancercell lines (CaSki and CUMC-6);

FIG. 4: the results of northern blot analyses for HCCR-1 gene expressedin normal lung tissue and lung cancer cell lines (NCI-H358, NCI-H460,NCI-H441, NCI-H1299, NCI-H520, NCI-H2009, and NCI-H157);

FIG. 5A: the results of northern blot analyses for HCCR-1 gene expressedin normal human 12-lane multiple tissues;

FIG. 5B: the results obtained with the same sample of FIG. 5A hybridizedwith β-actin;

FIG. 6A: the results of northern blot analyses for HCCR-1 gene expressedin human cancer cell lines;

FIG. 6B: the results obtained with the same sample of FIG. 6A hybridizedwith β-actin;

FIG. 7A: the results of northern blot analyses for HCCR-1 gene expressedin human tumor tissues and their normal counterparts;

FIG. 7B: the results obtained with the same sample of FIG. 7A hybridizedwith β-actin;

FIG. 8: a micrograph illustrating representative characteristics of insitu hybridized human cervical cancer tissues;

FIG. 9: a phase-contrast feature of monolayer-cultured wild type NIH/3T3cells;

FIG. 10: a phase-contrast feature of monolayer-cultured HCCR-1 cells;

FIG. 11: hematoxylin-eosin staining of monolayer-cultured HCCR-1 cells;

FIG. 12: a transmission electron micrograph illustrating representativecharacteristics of cultured HCCR-1 cells;

FIG. 13: tumorigenicity of HCCR-1 cells in nude mouse;

FIG. 14: hematoxylin-eosin staining of subcutaneous tumour nodules takenfrom nude mice;

FIG. 15: transmission electron micrographs illustrating representativecharacteristics of nude mice-derived subcutaneous tumor tissue;

FIG. 16: phase-contrast features of monolayer-cultured nude mice-derivedHCCR-1N cells;

FIG. 17: sodium dodecyl sulfate (SDS)-PAGE results showing proteinexpression patterns before and after the IPTG induction;

FIG. 18: the result of western blotting analysis of NIH/3T3 cellswithout transfection (wild type), NIH/3T3 transfected with pcDNA3 vectoralone (pcDNA3) and HCCR-1 cells;

FIG. 19: the result of western blotting analysis of human tumour tissuesof kidney, lung, ovary and cervix and their normal counterparts;

FIG. 20: the immunohistochemical study of HCCR-1-transfected NIH 3T3cells against reticulin fibers (×250);

FIG. 21: the expression of epithelial marker, keratin inHCCR-1-transfected NIH/3T3 cells (×250);

FIG. 22: the expression of epithelial membrane antigen inHCCR-1-transfected NIH/3T3 cells (×250);

FIG. 23: the expression of mesenchymal marker, vimentin inHCCR-1-transfected NIH/3T3 cells (×250);

FIG. 24: the PKC activities in NIH/3T3 cells without transfection(wild-type), NIH/3T3 transfected with pcDNA3 vector alone (pcDNA3) andNIH/3T3 transfected with HCCR-1 protoncogene (HCCR-1 cells);

FIG. 25: the telomerase activities in 293 cells, +RNase, NIH/3T3 cellswithout transfection (wild-type), NIH/3 T3 transfected with pcDNA3vector alone (pcDNA3) amd NIH/3T3 transfected with HCCR-1 protoncogene(HCCR-1 cells);

FIG. 26A: the results of aRT-PCR amplification of HCCR-1 cDNA in H-358lung carinoma cell lines treated with anti-sense oligodeoxynucleoties;

FIG. 26B: the results obtained with the same sample of FIG. 26Ahybridized with β-actin;

FIG. 27: growth curves of H-358 lung carcinoma cell lines treated withsense, missense or anti-sense HCCR-1 ODN, and untreated parental cells;

FIG. 28: HCCR-1 protein expressions in fetal 16-(F16), 18-(F18),20-(F20), postnatal 1-(P1), 7-(P7), 14-day (P14) and adult rat kidneytissue extracts;

FIG. 29: immunohistochemical staining of 20 day-old fetal rat kidney(×42); and

FIG. 30: differential-interference contrast microscopy of 18 day-oldfetal rat kidney illustrating HCCR-1 immunostaining in the basolateralplasma membrane of medullary collecting duct (×220).

DETAILED DESCRIPTION OF THE INVENTION

The novel protooncogene of the present invention, i.e., human cervicalcancer 1 (hereinafter “HCCR-1 protooncogene”), consists of 2118 basepairs and has the DNA sequence of SEQ ID NO:1.

In SEQ ID NO: 1, the full open reading frame corresponding to base Nos.9 to 1088 is a protein encoding region and the predicted amino acidsequence derived therefrom is shown in SEQ ID NO: 2 which consists of360 amino acids (“HCCR-1 protein”). Further, the region corresponding tobase Nos. 9 to 83 of SEQ ID NO: 1 encodes a signal peptide with thepredicted amino acid sequence of amino acid Nos. 1 to 25 in SEQ ID NO:2; and the region represented by nucleotide No. 435 to 494 of SEQ ID NO:1 encodes a single transmembrane domain having the predicted amino acidsequence of amino acid Nos. 143 to 162 of SEQ ID NO: 2. This suggeststhat the protooncogene of the present invention is a membrane-boundgene.

A single potential N-glycosylation site (corresponding to base Nos. 945to 953 of SEQ ID NO: 1 and amino acid Nos. 313 to 315 of SEQ ID NO: 2)is present at the C-terminal side of the HCCR-1 protein, which suggeststhat HCCR-1 is a type II membrane protein. The polyadenylation signalcorresponds to the nucleotide Nos. 2008-2012 of SEQ ID NO:1.

The predicted extracellular domain of HCCR-1 corresponds to base Nos.495-1088 with the predicted amino acid sequence of amino acid Nos.163-360 consisting of 198 amino acids with 5 cysteine residues. Thepredicted intracellular domain contains 117 amino acids (correspondingto nucleotide Nos. 84-434 of SEQ ID NO:1 and amino acid Nos. 26-142 ofSEQ ID NO:2) with two potential protein kinase C (PKC) phosphorylationsites at Ser-42 and Ser-48, and two potential N-myristylation sites atGly-34 and Gly-38. Further computer-assisted analyses indicate thatHCCR-1 is markedly hydrophobic and possesses a characteristic singlemembrane-spanning domain and pre-secretory signal peptide as shown inFIG. 2.

In consideration of the degeneracies of codons and the preferred codonsin a specific animal wherein the protooncogene of the present inventionis to be expressed, various changes and modifications of the DNAsequences of SEQ ID NO:1 may be made, e.g., in the coding area thereofwithout adversely altering the amino acid sequence of the expressedprotein, or in the non-coding area without adversely affecting theexpression of the protooncogene. Therefore, the present invention alsoincludes, in its scope, a polynucleotide having substantially the samebase sequence as the inventive protooncogene, and a fragment thereof. Asused herein, “substantially the same polynucleotide” refers to apolynuleotide whose base sequence shows 80% or more, preferably 90% ormore, most preferably 95% or more homology to the protooncogene of thepresent invention.

The protein expressed from the protooncogene of the present inventionconsists of 360 amino acids and has the amino acid sequence of SEQ IDNO: 2. The molecular weight of this protein is about 40 kDa. However,various substitution, addition and/or deletion of the amino acidresidues of protein may be performed without adversely affecting theprotein's function. Further, a portion of the protein may be used when aspecific purpose is to be fulfilled. These modified amino acids andfragments thereof are also included in the scope of the presentinvention. Therefore, the present invention includes, in its scope, apolypeptide having substantially the same amino acid sequence as theprotein derived from the oncogene of the present invention and afragment thereof. As used herein, “substantially the same polypeptide”refers to a polypeptide whose amino acid sequence shows 80% or more,preferably 90% or more, most preferably 95% or more homology to theamino acid sequence of SEQ ID NO:2.

The protooncogene, or the protein, of the present invention can beobtained from human cancer tissues or synthesized using a conventionalDNA or peptide synthesis method. Further, the gene thus prepared may beinserted to a conventional vector to obtain an expression vector, whichmay, in turn, be introduced into a suitable host, e.g., an E. coli oryeast cell, The cells transformed with a vector containing the HCCR-1protooncogene or a fragment thereof is hereinafter referred to a “HCCR-1cell”.

The transformed host may then be used in producing the inventive DNA orprotein on a large scale. For example, E. coli JM109 is transfected withHCCR-1 protooncogene by using pGEM-T easy vector and the JM109/HCCR-1was deposited on Oct. 11, 1999 with the Korean Collection for TypeCultures (KCTC)(Address: Korea Research Institute of Bioscience andBiotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333,Republic of Korea) under the accession number, KCTC 0667BP, inaccordance with the terms of Budapest Treaty on the InternationalRecognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

In preparing a vector, expression-control sequences, e.g., promoter.terminator, self replication sequence and secretion signal, are suitablyselected depending on the host cell used.

The overexpression of the protooncogene of the present invention occursnot in normal cervical and lung tissues but in cervical cancer tissues,cervical cancer cell lines and lung cancer cell lines. This suggeststhat the protooncogene of the present invention induces cervical andlung cancers. Further, when a normal fibroblast cell, e.g., NIH/3T3 cellline, is transfected with the protooncogene of the present invention, anabnormal cells is produced. Morphological characterizations with opticaland electronic microscopes show that the abnormal cell has the form of atumor cell.

When the normal fibroblast cell transfected with the protooncogene ofthe present invention is injected into the posterial lateral aspect of anude mouse, tumorigenesis is observed after about 21 days from theinjection, the tumor size becoming 1.5 cm×1.5 cm in 40 days. By usinghematoxylin-eosin dye method, it can be confirmed that the tumor cellsare cancerous. The formation of the epithelial carcinoma can also beconfirmed by using transmission electron microscopy andimmunhistochemical staining methods.

In addition to epithelial tissues such as cervical and lung cancertissues, the overexpression of the protooncogene of the presentinvention is also observed in various other cancer tumors such asleukemia, lymphoma, kidney, liver and ovarian cancers. Therefore, theprotooncogene of the present invention is believed to be a factor commonto all forms of various cancer and it can be advantageously used in thediagnosis of various cancers and the production of a transformed animalas well as in an anti-sense gene therapy.

A diagnostic method that can be performed using the protooncogene of thepresent invention may comprise, for example, the steps of hybridizingnucleic acids separated from the body fluid of a subject with a probecontaining the protooncogene of the present invention or a fragmentthereof, and determining whether the subject has the protooncogene byusing a conventional detection method in the art. The presence of theprotooncogene may be easily detected by labeling the probe with aradioisotope or an enzyme. Therefore, a cancer diagnostic kit containingthe protooncogene of the present invention or a fragment thereof is alsoincluded in the scope of the present invention.

A transformed animal produced by introducing the protooncogene of thepresent invention into a mammal, e.g., a rat, is also included in thescope of the present invention. In producing such a transformed animal,it is preferred to introduce the inventive protooncogene to a fertilizedegg of an animal before the 8th cell cycle stage. The transformed animalcan be advantageously used in screening for carcinogens or anticanceragents such as antioxidants.

The present invention also provides an anti-sense gene which is usefulin a gene therapy. As used herein, the term “an anti-sense gene” means apolynucleotide comprising a base sequence which is fully or partiallycomplementary to the sequence of the mRNA which is transcribed from theprotooncogene having the base sequence of SEQ ID NO:1 or a fragmentthereof, said nucleotide being capable of preventing the expression ofthe open reading frame (ORF) of the protooncogene by way of attachingitself to the protein-binding site of mRNA.

An example of the anti-sense gene of the present invention is a 18-merHCCR-1 anti-sense oligodeoxinucleotide (ODN) having the base sequence ofSEQ ID NO:3. Therefore, the present invention also includes, in itsscope, a polynucleotide comprising substantially the same base sequenceas SEQ ID NO:3 and a fragment thereof.

The present invention also includes within its scope a process fortreating or preventing cancer in a subject by way of administering atherapeutically effective amount of the inventive anti-sense genethereto.

In the inventive anti-sense gene therapy, the anti-sense gene of thepresent invention is administered to a subject in a conventional mannerto prevent the expression of the protooncogene. For example, theanti-sense ODN is mixed with a hydrophobized poly-L-lysine derivative byelectrostatic interaction in accordance with the method disclosed byKim, J. S. et al. (J. Controlled Release, 53, 175-182 (1998)) and theresulting mixed anti-sense ODN is administered intravenously to asubject.

The present invention also includes within its scope an anti-cancercomposition comprising the anti-sense gene of the present invention asan active ingredient, in association with pharmaceutically acceptablecarriers, excipients or other additives, if necessary. Thepharmaceutical composition of the present invention is preferablyformulated for administration by injection.

The amount of the anti-sense gene actually administered should bedetermined in light of various relevant factors including the conditionto be treated, the chosen route of administration, the age and weight ofthe individual patient, and the severity of the patient's symptoms.

The protein expressed from the inventive protooncogene may be used inproducing an antibody useful as a diagnostic tool. The antibody of thepresent invention may be prepared in the form of a monoclonal orpolyclonal antibody in accordance with any of the methods well known inthe art by using a protein having the amino acid sequence of SEQ ID NO:2or a fragment thereof. Cancer diagnosis may be carried out using any ofthe methods known in the art, e.g., enzyme linked immunosorbentassay(ELISA), radioimmunoassay (RIA), sandwich assay, western blot orimmunoassay blot on polyacrylic gel, to asses whether the protein isexpressed in the body fluid of the subject. Therefore, a cancerdiagnostic kit containing the protein having the amino acid sequence ofSEQ ID NO:2 or a fragment thereof is also included in the scope of thepresent invention.

A continuously viable cancer cell line may be established by using theprotooncogene of the present invention, and such a cell line may beobtained, for example, from tumor tissues formed on the back of a nudemouse by injecting fibroblast cells transformed with the protooncogeneof the present invention. The cell lines thus prepared may beadvantageously used in searching for anti-cancer agents.

The following Examples and Test Examples are given for the purpose ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Cultivation of Tumor Cells and Separation of Total RNA

Step 1-1: Cultivation of Tumor Cells

For differential display of mRNA, normal cervical tissues, untreatedprimary cervical cancer tissues and metastatic common iliac lymph nodetissues were obtained from cervical cancer patients who underwentradical hysterectomy. The human cervical cancer cell line used in thedifferential display method was CUMC-6 cell line described by Kim etal., (Gynecol. Oncol., 62: 230-240 (1996)).

Cells from the above-described tissues and CUMC-6 were maintained onWaymouth's MB 752/1 medium (Gibco) supplemented with 2 mmol/L ofglutamine, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 10%of fetal bovine serum (Gibco). Only the cell suspensions with greaterthan 95% viability, as assessed by trypan blue dye exclusion describedby Freshney (“Culture of Animal Cells: A Manual of Basic Technique” 2ndEd., A. R. Liss, New York (1987)) were used in the present experiments.

Step 1-2: Isolation of Total RNA and Differential Display of mRNA

Total RNAs were extracted from normal cervical tissues, primary cervicalcancer tissues, metastatic common iliac lymph node tissues and CUMC-6cells obtained in Step 1-1 using a commercial system (RNeasy total RNAkit) provided by Qiagen (Qiagen Inc., Germany) and the removal of DNAcontaminants from the RNAs was accomplished using Message clean kit(GenHunter Corp., Brookline, Mass.).

EXAMPLE 2 Differential Display Reverse Transcription (DDRT)-PCR

Differential display reverse transcription was performed in accordancewith the reverse transcription-polymerase chain reaction (RT-PCR) methoddescribed by Liang and Pardee (1992), supra, with minor modifications.

First, reverse transcription was carried out using 0.2 μg each of thetotal RNAs obtained in Step 1-2 of Example 1 and one of the threeprimers, i.e., H-T11G, H-T11C, or H-T11A, as anchored oligo-dT primers(RNAimage kit, GenHunter Cor., Massachusetts, USA).

Then PCR was conducted using the same anchored primers and one of thearbitrary 5′ 13 mer (RNAimage primer sets 1-4, H-AP 1-32) in thepresence of 0.5 mM [α-³⁵S]-labeled dATP (1200 Ci/mmol). The PCR thermalcycle was repeated 40 times, the cycle being composed of: 95° C. for 40sec., 40° C. for 2 min. and 72° C. for 40 sec., and finally the reactionwas carried out at 72° C. for 5 min.

PCR-amplified fragments were resolved in 6% polyacrylamide sequencinggels. Differentially expressed fragments were identified by inspectionof autoradiograms.

Bands of more than 200 base pairs, CC214, were excised from the driedsequencing gel. The CC214 cDNAs were eluted by boiling for 15 min andreamplified with the same primer pairs and PCR conditions as used in theabove amplification step except that no [α-³⁵S]-labeled dATP and 20 μMdNTPs were used.

EXAMPLE 3 Cloning

The reamplified CC214 PCR product obtained as above was inserted intothe pGEM-T Easy vector using an TA cloning system (Promega, USA) inaccordance with the manufacturer's instructions.

Step 3-1: Ligation

2 μl of the reamplified CC214 PCR product obtained in Example 2, 1 μl ofpGEM-T easy vector (50 ng), 1 μl of T4 DNA ligase 10× buffer solutionand 1 μl of T4 DNA ligase (3 weiss units/μl; T4 ligase, Promega, USA)were charged into a 0.5 ml tube and distilled water was added thereto toa final volume of 10 μl. The ligation reaction mixture was incubatedovernight at 14° C.

Step 3-2: TA Cloning Transformation

TA cloning transformation was performed using the following protocol.

E. coli JM109 (Promega, Wisconsin, USA) was incubated in 10 ml of LBbroth (Bacto-trypton 10 g, Bacto-yeast extract 5 g, NaCl 5 g) until theoptical density at 600 nm reached about 0.3 to 0.6. The cultured mixturewas kept at 0° C. for 10 minutes and centrifuged at 4000 rpm at 4° C.for 10 minutes. The supernatant was removed and cells were harvested.The harvested cell pellet was exposed to 10 ml of 0.1M CaCl₂ at 0° C.for 30 minutes to 1 hour to obtain competent cells. The resultant wascentrifuged at 4000 rpm at 4° C. for another 10 minutes and thecollected cells were suspended in 2 ml of 0.1M CaCl₂ at 0° C.

200 μl of the competent cell suspension was transferred to a microfugetube and 2 μl of the ligation product obtained in Step 3-1 was addedthereto. The mixture was incubated in a water bath at 42° C. for 90seconds and rapidly cooled to 0° C. Added thereto was 800 μl of SOCmedium (Bacto-trypton 2.0 g, Bacto-yeast extract 0.5 g, 1M NaCl 1 ml, 1MKCl 0.25 ml, TDW 97 ml, 2M Mg²⁺ 1 ml, 2M glucose 1 ml) and the mixturewas incubated at 37° C. for 45 minutes at 220 rpm in a rotary shakingincubator.

LB agar plates, containing ampicillin (50 ul/ml) were prepared byspreading 25 μl of X-gal (40 mg/ml stock in dimethylformamide) on top ofagar with a glass spreader. 25 μl of the transformed cells thus obtainedwas spread thereon and the plates were incubated at a 37° C. incubatorovernight. White colonies were loaded on an LB agar plate containingampicillin and transformed E. coli, i.e., JM109/CC214 were selected andincubated in a terrific broth (TDW 900 ml, Bacto-trypton 12 g,Bacto-yeast extract 24 g, glycerol 4 ml, 0.17M KH₂PO₄, 0.72 N K₂HPO₄ 100ml).

EXAMPLE 4 Separation of Recombinant Plasmid DNA

The CC214 DNA of the transformed E. coli was separated by employingWizard™ Plus Minipreps DNA Purification Kit (Promega, USA) in accordancewith the manufacturer's instructions.

A portion of the plasmid DNA thus separated was treated with ECoRIenzyme and subjected to gel electrophoresis to confirm the insertion ofCC214 partial sequence in the plasmid.

EXAMPLE 5 Sequence Analysis of DNA

The CC214 PCR product obtained in Example 2 was subjected to PCR inaccordance with the conventional method and the cloned, reamplifiedCC214 PCR fragments were subjected to sequence analysis according to thedideoxy chain termination method using a Sequenase version 2.0 DNAsequencing kit (United states Biochemical, Cleveland, Ohio) inaccordance with the manufacturer's instructions.

The base sequence of the DNA corresponds to nucleotide Nos. 1883-2088 inSEQ ID NO:1 and is designated “CC214”.

The differential display reverse transcription polymerase chain reaction(DDRT-PCR) of the 206 bp cDNA fragment, i.e., CC214 obtained above wascarried out using a 5′ arbitrary primer H-AP21 and a 3′ H-T11C anchoredprimer and resolved by electrophoresis. Identification of altered geneexpression by DD in the primary cervical cancer, metastatic lymph nodetissue and CUMC-6 cells is shown in FIG. 1. As can be seen in FIG. 1,the 206 bp cDNA fragment, i.e., CC214 was expressed in the cervicalcancer, metastatic tissue and CUMC-6 cervical cancer cells but not inthe normal tissue.

EXAMPLE 6 Full Length cDNA Sequence Analysis of the HCCR-1 Protooncogene

A bacteriophage λ gt11 human lung embryonic fibroblast cDNA library (seeMiki, T. et al., Gene, 83:137-146 (1989)) was screened by plaquehybridization with ³²P-labeled CC214 as a probe. The full-length HCCR-1cDNA clone, containing a 2118 bp insert in pCEV-LAC vector was obtainedfrom the human lung embryonic fibroblast cDNA library and registered atGenBank on Nov. 5, 1999 under the accession number AF195651.

HCCR-1 clone inserted into λpCEV vector (see Miki, T. et al., supra) wasexcised out of the phage in the form of the ampicilline-resistantpCEV-LAC phagemid vector (see Miki, T. et al., supra) by Not I cleavage.

To make a HCCR-1 plasmid DNA, pCEV-LAC vector containing HCCR-1 gene wasligated with T4 DNA ligase and ligated clone was transformed into E.coli JM109.

The transformed E. coli JM109/HCCR1 thus obtained was deposited on Oct.11, 1999 with the Korean Collection for Type Cultures (KCTC)(Address:Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52,Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under theaccession number, KCTC 0667BP, in accordance with the terms of BudapestTreaty on the International Recognition of the Deposit of Microorganismfor the Purpose of Patent Procedure.

The full sequence of HCCR-1 consists of 2118 bp which is identified inSEQ ID NO:1.

In SEQ ID NO:1, the full open reading frame of the HCCR-1 protooncogeneof the present invention corresponds to nucleotides No. 9 to 1088 and ispredicted to encode amino acid sequence shown in SEQ ID NO:2 whichconsists of 360 amino acids. Further, the region corresponding tonucleotide Nos. 9 to 83 of SEQ ID NO:1 encodes a signal peptide having25 amino acids corresponding to amino acid Nos. 1 to 25 of SEQ ID NO:2;the region of nucleotide Nos. 435 to 494 of SEQ ID NO:1 encodes a singletransmembrane domain whose amino acid sequence corresponds to amino acidNos. 143 to 162 in SEQ ID NO:2. This indicates that the protooncogene ofthe present invention is a membrane-bound gene.

A single potential N-glycosylation site (corresponding to base Nos. 945to 953 of SEQ ID NO: 1 and amino acid Nos. 313 to 315 of SEQ IS NO: 2)is present at the C-terminal side of the HCCR-1 protein, which suggeststhat HCCR-1 is a type II membrane protein. The polyadenylation signalcorresponds to the nucleotide Nos. 2008-2012 of SEQ ID NO:1.

The predicted extracellular domain contains 198 amino acids with 5cysteine residues. The predicted intracellular domain contains 117 aminoacids (corresponding to nucleotide Nos. 84-434 of SEQ ID NO:1 and aminoacid Nos. 26-142 of SEQ ID NO:2) with two potential PKC phosphorylationsites at Ser-42 and Ser-48, and two potential N-myristylation sites atGly-34 and Gly-38. Further computer-assisted analyses indicated thatHCCR-1 is markedly hydrophobic and possesses a characteristic singlemembrane-spanning domain and pre-secretory signal peptide as shown inFIG. 2. In FIG. 2, the X-axis represents the amino acid sequence numberof the peptide of the present invention and the Y-axis, thehydrophobicity of the peptide.

EXAMPLE 7 Northern Blot Analysis of the HCCR-1 Gene in Various Cells

Total RNAs were extracted from various tissues and cell lines as inExample 1.

To determine the level of HCCR-1 gene expression, 20 μg denatured totalRNAs from each tissue or cell lines were electrophoresed through 1%formaldehyde agarose gel and transferred to nylon membranes(Boehringer-Mannheim, Germany). The blots were hybridized with a³²P-labeled random-primed HCCR-1 full cDNA probe which was preparedusing a rediprime II random prime labeling system (Amersham, England).The northern blot analysis was repeated twice and the results werequantified by densitometry and normalized with β-actin.

FIG. 3 shows the results of northern blot analyses for HCCR-1 geneexpressed in normal cervical tissues, cervical cancer tissues andcervical cancer cell lines (CaSki and CUMC-6). As can be seen in FIG. 3,the transcription level of HCCR-1 is high in the cervical cancer tissuesand cancer cell lines (CaSki(ATCC CRL 1550) and CUMC-6), but very low orundetactable in the normal cervical tissues.

FIG. 4 shows the results of northern blot analyses for HCCR-1 geneexpressed in normal lung tissues and seven lung cancer cell lines, i.e.,H358 (ATCC NCI-H358), H460 (ATCC NCI-H460), H441 (ATCC NCI-H441), H299(ATCC NCI-H299), H520 (ATCC NCI-H520), H2009 (ATCC NCI-H2009), and H157(ATCC NCI-H157). As shown in FIG. 4, HCCR-1 transcription level in highlevel in lung cancer cell lines H358, H460, H1299, H520, and H157, butnot detectable in the normal lung tissues.

FIG. 5A shows the results of northern blot analyses for HCCR-1 geneexpressed in normal human 12-lane multiple tissues; brain, heart,skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine,placenta, lung and leukocyte tissues (Clontech). FIG. 5B shows theresults obtained with the same samples hybridized with a β-actin probeto confirm mRNA integrity. As can be seen in FIG. 5A, HCCR-1 mRNA (˜2.1kb) is weakly present or absent in many normal tissues, but the level ofexpression was high in normal kidney tissue.

FIG. 6A shows the results of northern blot analyses for HCCR-1 geneexpressed in human cancer cell lines; HL-60, HeLa, K-562, MOLT-4, Raji,SW480, A549 and G361 (Clontech). Fog. 6B shows the results obtained withthe same samples hybridized with a β-actin probe to confirm mRNAintegrity. As can be seen in FIG. 6A, HCCR-1 is transcribed at a highlevel in the human leukaemia and lymphoma cell lines such as chronicmyelogenous leukaemia K-562, Burkitt's lymphoma Raji, lymphoblasticleukaemia MOLT-4 and promyelocytic leukaemia HL-60 as well as HeLacells.

K-562, MOLT-4 and HL-60, in particular, show higher transcription levelsas compared with normal leukocyte by factors of approximately 190, 90and 70, respectively. HCCR-1 expression levels in colorectal cancerSW480, lung cancer A549 and melanoma G361 cell lines are lower thanthose of leukemia and lymphoma.

Further, northern blotting analyses of the human kidney, liver, lung,ovary and cervix tumor tissues and their normal counterparts werecarried out. As shown in FIG. 7A, HCCR-1 was transcribed at a high levelin the human cancer cells, while the expression of HCCR-1 gene is barelyobservable in the normal cells. FIG. 7B shows the results obtained withthe same samples hybridized with β-actin probe to confirm mRNAingegrity.

EXAMPLE 8 Micrograph of in situ Hybridized Human Cervical Cancer Tissues

For in situ hybridization, human cervical cancer tissue was fixed inperiodate-lysine-paraformaldehyde, embedded in a wax according to theprocedure described by Ahn et al. (Am. J. Physiol. 265, F792-F801(1993)) and sectioned (5 μm). A full-length HCCR-1 cDNA fragment wasused to synthesize a digoxigenin-labelled RNA probe. RNA in situhybridization was carried out with the anti-sense RNA probe which wasprepared using a DIG RNA Labelling Kit (Boehringer Mannheim). The senseRNA probe was used as a negative control.

FIG. 8 shows a micrograph illustrating representative characteristics ofin situ hybridized human cervical cancer tissues. As can be seen in FIG.8, the in situ hybridized cervical cancer tissues are confirmed tocontain a high-level of HCCR-1 gene. No staining was detected in thesurrounding normal fibrous tissues.

EXAMPLE 9 Construction of Expression Vectors and Transformation of Cells

Step 9-1: Preparation of a Vector Containing HCCR-1

An expression vector containing the coding region of HCCR-1 wasconstructed as follows.

First, the entire HCCR-1 cDNA obtained in Example 6 was inserted intothe SalI restriction site of a prokaryotic expression vector, pCEV-LAC(see Miki, T. et al., Gene, 83: 137-146 (1989)). Then, the SalI fragmentwas isolated from the pCEV-LAC/HCCR-1 vector.

Then, pcDNA3 (Invitrogen) was digested with XhoI to make a compatibleend with SalI. The SalI fragment containing the full length HCCR-1coding sequence was inserted into the XhoI-digested pcDNA3.Lipofectamine (Gibco BRL) was used to introduce the resultingpcDNA3/HCCR-1 expression vector into NIH/3T3 cells (ACTC CRL, 1658,USA), followed by selection in a medium supplemented with G418 (Gibco).The resulting NIH/3T3 cells transfected with HCCR-1 was designated“HCCR-1 cells”. Another population of NIH/3T3 cells containing pcDNA3alone was prepared as a control and designated “pcDNA3 cells”.

Step 9-2: NIH/3T3 Fibroblast Cells Transfected with the HCCR-1Protooncogene

The wild type normal NIH/3T3 cell, a differentiated fibroblast cellline, is a spindle shaped cell having a long slender nucleus and ascanty amount of cytoplasm as shown in FIG. 9. When HCCR-1 was expressedin the NIH/3T3 expressing HCCR-1 (HCCR-1 cells) obtained in Step 9-1,the cell shape changes into a polygonal form with an ovoid nucleus andplump cytoplasm, as shown in FIG. 10.

Monolayer cultured HCCR-1-transfected NIH/3T3 cells which is stainedwith hematoxylin-eosin, exhibit nuclear pleiomorphism, distinctnucleoli, granular chromatin patterns, tumor giant cells and atypicalmitotic figures as shown in FIG. 11.

For transmission electron microscopy (TEM), the cells and tissues werefixed with 2.5% glutaraldehyde in a phosphate buffer (pH 7.4). They werethen postfixed with a 2% osmium tetroxide. Specimens were dehydrated ina graded series of ethanols and embedded in Epon 812. Ultrathin sectionsthereof were stained with uranyl acetate and lead citrate, andphotographed by TEM (JEOL 1,200 EX, Tokyo, Japan).

The TEM picture shown in FIG. 12 reveals that cultured tumour cells havemicrovilli and well-developed organelles (inset). As can be seen in FIG.12, the HCCR-1 cell has microvilli on the cell surface, lobulatednucleus with prominent nucleoli and well-developed rough endoplasmicreticula (rER) and Golgi complexes (circle). In FIG. 12, the scale barcorresponds to 3 μm. In higher magnification of the area indicated bycircle (inset), the scale bar corresponds to 1 μm.

EXAMPLE 10 Tumorigenicity and Metastasis of HCCR-1 Protooncogene inAnimal

To analyze tumourigenicity, 5×10⁶ HCCR-1 cells were injectedsubcutaneously into the posterior lateral aspect of the trunk of 9 mice(5-week-old athymic nu/nu on BALB/c background). Nude mice weresacrificed when the subcutaneous tumors reached 1.5-2.5 cm in diameter.

All 9 mice injected with HCCR-1 cells showed palpable tumors after 21days as shown in FIG. 13.

Nude mice bearing HCCR-1 allografts display characteristics of anepithelial carcinoma. FIG. 14 shows hematoxylin-eosin staining ofsubcutaneous tumor nodules taken from the nude mice. The sections of thetumour nodules revealed typical epithelial cell nests separated byfibrous stroma.

EXAMPLE 11 Electron Microscopy of HCCR-1 Protooncogene—Induced TumorTissue and Establishment of New Cancer Cell Line

Tumor tissues taken from the tumor nodules formed on the nude mouse ofExample 10 were examined with an electron microscope, which revealedthat tumor nodules showed well-developed organelles and tumour cells areconnected by desmosomes (FIG. 15). As shown in FIG. 15, the tumor tissueconsists of tightly adhered cells with intercellular junction (circle).In FIG. 15, the scale bar corresponds to 3 μm. In higher magnificationof the area indicated by circle illustrating desmosome (inset), thescale bar corresponds to 0.5 μm.

The cells obtained from the above tumour tissue was cultured in aconventional manner using 20% fetal bovine serum and the cultured cellswere designated HCCR-1N cells which have cytological features similar toHCCR-1 cells in vitro as shown in FIG. 16.

EXAMPLE 12 Determination of Size of Protein Expressed after theTransfection of E. coli with HCCR-1 Protooncogene

A portion of HCCR-1 protooncogene corresponding to nucleotide Nos.123-473 and predicted amino acid Nos. 39-155 was inserted into themultiple cloning site of pET-32b(+) vector (Novagen) and the resultingpET-32b(+)/HCCR-1 vector was transfected into E. coli BL21 (ATCC 47092).The transfected E. coli was incubated using an LB broth medium in arotary shaking incubator, diluted by 1/100, and incubated for 3 hours. 1mM isopropyl β-D-thiogalacto-pyranoside (IPTG, Sigma) was added theretoto induce the protein synthesis.

The E. coli cells in the culture were disrupted by sonication andsubjected to gel electrophoresis using 12% sodium dodecyl sulfate (SDS)before and after the IPTG induction. FIG. 17 shows the SDS-PAGE resultswhich exhibit a protein expression pattern of the E. coli BL21 straintransfected with pET-32b(+)/HCCR-1 vector. After the IPTG induction, asignificant protein band was observed at about 35 kDa. This 35 kDa fusedprotein contained an about 20 kDa Trix•Tag thioredoxin protein expresseda the gene in pET-32b(+) vector.

EXAMPLE 13 Production of Antibody

The 35 kDa fused protein isolated from the E. coli BL21 straintransfected with pET-32b(+)/HCCR-1 vector in Example 12 was purified byusing a His-Bind Kit (Novagen). Immunoblotting of the purified peptideconfirmed the presence of a major amount of a 35 kDa protein.

Then, two 6-seek old Sprague-Dawley rats each weighing about 150 g wereeach subcutaneously immunized with 1 mg of the peptide thus obtained,weekly for 3 times. Blood samples were obtained from the immunized ratsand centrifuged to obtain a polyclonal serum. The anti-HCCR-1 activityof the polyclonal serum was determined and confirmed by enzyme-linkedimmunosorbent assay (1:10,000)

EXAMPLE 14 Immunoblot Confirming Antibody Specificity

For western blot analysis, those cells identified in FIGS. 18 and 19were harvested and lysed in a Laemmli sample buffer in accordance withthe method described by Laemmli (Nature 227: 680-685 (1970)). Thecellular proteins were separated by 10% SDS-PAGE and then electroblottedonto nitrocellulose membranes. The membranes were incubated with the ratpolyclonal anti-HCCR-1 serum prepared in Example 13 for 16 h. Afterwashing, the membranes were incubated with a blocking solutioncontaining 1:1,000 dilution of peroxidase-conjugated goat anti-ratimmunoglobulin (Jackson ImmunoResearch) as a secondary antibody.Proteins were revealed by an ECL-Western blot detection kit (Amersham).

As shown in FIG. 18, HCCR-1 protein is overexpressed in HCCR-1 cells,while only faint bands are observed for the wild type and cellstransfected with the vector alone (pcDNA3). This result illustrates thespecificity of the anti-HCCR-1 antibodies in the polyclonal serum.

Further, the HCCR-1 antibody in the polyclonal serum recognizedapproximately 40 kDa protein in human protein extracts from differenttissues. As shown in FIG. 19, human tumor tissues including carcinomasof the kidney, lung, ovary and cervix showed increased HCCR-1 proteinexpression when compared with their normal counterparts.

EXAMPLE 15 Immunohistochemistry

The tumor nodules formed on the nude mouse of Example 9 were incubatedwith anti-vimentin, anti-keratin, anti-EMA (epithelial membrane antigen)antibodies (DAKO) and polyclonal antibody raised against HCCR-1,respectively. Then, immunohistochemistry was carried out on 5μm-cryosections of the incubated tumor nodules.

Binding of primary antibody was visualized by biotinylated secondaryantibody, avidin, biotinylated horseradish peroxidase and AEC(Aminoethyl Carbaxzole Substrate Kit) as the chromogen (HISTOSTAIN-BULKKITS, Zymed). The immunohistochemical study revealed that HCCR-1transfection into NIH/3T3 cells caused the conversion of the cell naturefrom mesenchymal to epithelial. The cell nests were enveloped byreticulin fibers as shown in FIG. 20.

The cells showed coexpression of epithelial markers, such as keratin(FIG. 21) and epithelial membrane antigen (FIG. 22) and of themesenchymal marker, vimentin (FIG. 23).

EXAMPLE 16 Protein Kinase C and Telomerase Activity Assays

To ensure that HCCR-1 modulates the protein kinase C (PKC) activity incells, PKC assay was performed using wild-type NIH/3T3 cells,pcDNA3-containing NIH/3T3 cells and HCCR-1-transfected NIH/3T3 cellsprepared in Step 9-1 of Example 9.

PKC activity was measured using the SignaTEC™ Protein Kinase C AssaySystem (Promega) according to the manufacturer's instructions. PKCactivity was defined as the difference of the amounts of PKCincorporated into substrate per minute in the absence and presence ofphospholipids. Each value is the means±s.d. of three independentexperiments.

The result in FIG. 24 shows that the PKC activity of HCCR-1-transfectedNIH/3T3 cells is about 10-fold higher than the wild-type.

To explain the tumorigenesis of HCCR-1, telomerase activities inwild-type NIH/3T3 cells, pcDNA3-containing NIH/3T3 cells andHCCR-1-transfected NIH/3T3 cells prepared in Step 9-1 of Example 9 weremeasured using the telomerase PCR-ELISA kit (Boehringer Mannheim)according to the manufacturer's instructions. Human telomerase-positiveimmortalized human kidney cells (293 cells) provided in the kit wereused as a positive control.

Used as a negative control was the 293 cells pretreated with RNase(+RNase). Assays were performed with an extract amount equivalent to1×10³ cells.

Results in FIG. 25 show the average mean optical density (OD) valuesfrom four separate experiments (means±s.d.). Consistent with theprevious study (Holt, S. E., Wright, W. E. and Shay J. W. Mol. CellBiol. 16, 2932-2939 (1996), wild-type NIH/3T3 cells showed detectabletelomerase activity. HCCR-1 gene transfection raised the telomeraseactivity by a factor of about 7 as compared with the wild-type cells.The high telomerase activity of the 293 cells was nullified bypretreatment with RNase.

EXAMPLE 17 Cell Cycle Experiments

Wild-type and HCCR-1-transfected NIH/3T3 cells cultured in a DMEM mediumat mid-log phase were growth arrested by incubation in a DMEM mediumcontaining 0.5% bovine calf serum for 36 h. Cells to be analyzed for theDNA content were harvested following trypsinization, and fixed in 70%ethanol. Fixed cells were then stained with propidium iodide asdescribed by Hedley (Flow Cytometry, DNA Analysis from Paraffin-embeddedBlocks; Darzynkiewicz, Z. & Crissman, H. A. eds., Academic Press, SanDiego, 1990).

First, 50 μg/ml of a propidium iodide staining solution (Sigma) and 100units per ml of RNase A (Boerhinger Mannheim) were added to 2×10⁶ cells.After incubation for 1 h, the cellular DNA content was determined byfluorescence analysis at 488 nm using a FACS Caliber (Becton Dickinson).A minimum of 1×10⁴ cells per sample was analyzed with Modfit 5.2software.

In order to study whether there was an alteration in the growthproperties of HCCR-1-transfected NIH/3T3 cells, cell cycle profiles wereexamined. The cell contents of the wild type NIH/3T3 cells and HCCR-1transfected NIH/3T3 cells (mid-log phase) in G₀/G₁, S, G₂/M phases weremeasured and the results are shown in Table I. TABLE I Wild Type HCCR-1Cell G₀/G₁ S G₂/M G₀/G₁ S G₂/M Cell 55.7 20.6 24 46.6 31.5 22.4Content(%)

As can be seen from Table I, the percentage of wild-type and HCCR-1transfected NIH/3T3 cells in the S-phase was 20.6% and 31.5%,respectively (mid-log phase). These results suggest that there was asignigicant shift of the cell pipulation out of the G₀/G₁-phase into theS-phase in HCCR-1 transfected NIH/3T3 cells.

To assess the serum-dependent cell cycle progression, cells werecultured in 0.5% bovine calf serum for 36 h. After incubation, cellswere released with 20% bovine calf serum and harvested at indicatedtimes. The cell contents of wild type NIH/3T3 cells and HCCR-1transfected NIH/3T3 cells in G₀/G₁, S, G₂/M phases at indicated timeswere measured and the results are shown in Table II. TABLE II CellContent (%) Wild Type HCCR-1 Cell Time (h) G₀/G₁ S G₂/M G₀/G₁ S G₂/M 077 8.0 14.9 70 21.8 8.7 12 72.2 14 14.2 66.9 24.0 9.6 24 49.6 13.4 37.256.7 24.7 19.2 48 58.3 18.3 23.7 52.7 30.4 17.5

As can be seen from Table II, few cells remained in the S-phase inwild-type cells measured at 0 h (8%). In contrast, a considerable numberof HCCR-1 cells measured at 0 h were still in the S-phase (21.8%),suggesting that constitutive overexpression of HCCR-1 allowed for arelative amount of resistance to serum deprivation-induced G₀/G₁ arrest.Following the release of cells from the growth arrest caused byserum-deprivation, there were consistent increases of over 10% in theS-phase populations of HCCR-1 cells as compared to wild-type cells atmeasured time intervals (12 h, 24 h and 48 h). Therefore, overexpressionof HCCR-1 could deregulate cell growth by shortening the G₀/G₁-phase andincreasing the S-phase population of cells.

EXAMPLE 18 Construction of Anti-Sense Oligodeoxinucleotide

Anti-sense and sense phosphorothioate oligodeoxynucleotide (ODNs)targeting the translation starting site of HCCR-1 mRNA were synthesizedbased on the human HCCR-1 cDNA sequence (GenBank accession numberAF195651) by cyanoethylphosphoramidite chemistry on an automated DNAsynthesizer (Expedite Nucleic Acid System, Framingham, Mass.).

The sequence of 18-mer HCCR-1 anti-sense ODN was5′-CCTGGACATTTTGTCACC-3′ (SEQ ID NO: 3; corresponding to nucleotide Nos.66 to 83 of SEQ ID NO:1). The corresponding sense sequence,5′-GGTGACAAAATGTCCAGG-3′(SEQ ID NO: 4), and missense sequence,5′-CGCGGATATTTCCTCACC-3′(SEQ ID NO: 5) were used as controls.

EXAMPLE 19 Cancer Gene Therapy Using HCCR-1 Antisense ODN

Step 19-1: Inhibition of Gene Expression

Exponentially growing 2×10⁵ H-358 lung carcinoma cells (ATCC CRL-5807)were detached by trypsin-EDTA and seeded in a 24-well plate.Lipofectamine (Gibco BRL) was used for oligodeoxynucleotide (ODN)treatment. Lipofectamine (5 μl/ml medium) was incubated with anappropriate amount of ODN to achieve a final concentration of 100 nMODN, in the cell suspension for 30 minutes at room temperature. Then1,000 μl portions of the mixture were added directly to the cells on the24-well plates and incubated for 1, 2, 3, 5 and 7 days, respectively.There was no cytotoxicity of the transfection reagent as controlled bytrypan blue dye exclusion assay.

To observe the inhibitory effect of HCCR-1 anti-sense ODN, the culturedlung carcinoma cells were treated with 100 nM each of sense, missenseand anti-sense ODNs obtained in Example 18, respectively. Inhibition ofHCCR-1 expression in anti-sense ODN-treated lung carcinoma cells wasdemonstrated by reverse transcription-polymerase chain reaction(RT-PCR). The sequences of oligonucleotide primers used for RT-PCR,synthesized according to the coding region of HCCR-1 cDNA were asfollows: forward, 5′-GGGAGATGGAGCATTTGAGA-3′ (SEQ ID NO:6, correspondingto nucleotides Nos. 376-395 of SEQ ID NO:1) and reverse,5′-GCTTCCGGAAAGCATGATAG-3′ (SEQ ID NO:7, corresponding to nucleotides554-573 of SEQ ID NO:1.

The dose of anti-sense exerting inhibitory effect was related to thelevels of HCCR-1 mRNA expression (FIG. 26A). 497 bp β-actin was used asan internal control to confirm mRNA integrity (FIG. 26B). A negativecontrol (N) contained nuclease-free water instead of RNA template.1000-bp ladder DNA size marker (M) was also used. As shown in FIG. 26A,the level of 198 bp HCCR-1 RT-PCR product decreased in a time-dependentmanner by anti-sense ODN treatment. HCCR-1 gene expression wascompletely inhibited in cells treated with 100 nM of anti-sense HCCR-1ODN for 7 days. In contrast, expected 198 bp HCCR-1 RT-PCR product wasdetected in cells treated with 100 nM of sense or missense HCCR-1 ODN,respectively, and in the untreated parental cells.

These results show that the treatment with 100 nM of anti-sense HCCR-1ODN completely blocks HCCR-1 gene expressions in lung carcinoma cells.

Step 19-2: Inhibition of Cell Growth

The growth phenotype of H-358 lung cancer cells treated with 100 nM ofsense, anti-sense or missense HCCR-1 oligodeoxynucleotide was assessedby growth curve.

In three independent experiments, H-358 lung cancer cells weretrypisinized and plated in the presence of 100 nM of sense, anti-senseor missense HCCR-1 ODN obtained in Example 18, and a growth medium(RPMI-1640) containing 100 nM of HCCR-1 ODN was replaced every otherday. Cells in triplicate dishes were detached and viable cells werecounted every other day using trypan blue dye exclusion.

As shown in FIG. 27, until 1 day of treatment, there were no discernabledifferences in cell growth among sense ([), missense (□) or anti-sense(o) HCCR-1 ODN-treated carcinoma cells. However, after 3 days of HCCR-1ODN treatment, anti-sense HCCR-1 ODN inhibited lung carcinoma cellgrowth in a time-dependent manner.

After 7 days exposure to antisense HCCR-1 ODN, the extent of growthinhibition was about 100% for H-358 lung carcinoma cells, while cellsexposed to sense or missense HCCR-1 ODN showed growth patterns similarto that of untreated wild-type H-358 cells (control cells,).

EXAMPLE 20 HCCR-1 Gene as a Regulator of Embryonic Kidney Development

Because the acquisition of epithelial properties by thefibroblast-derived HCCR-1 cells mimics the mesenchymal to epithelialconversion of cells during the organogenesis of the kidney (Giordano, S.et al., Proc. Natl. Acad. Sci. USA 90, 649-653 (1993): Tsarfaty, I., etal., Science 263, 98-101 (1994)), an experiment was conducted to examinewhether HCCR-1 is expressed in a developing kidney.

Total proteins in tissue extracts of fetal 16-, 18- and 20-day ratkidneys, postnatal 1-, 7- and 14-day rat kidneys and adult rat kidneywere subjected to SDS-PAGE. HCCR-1 protein in HCCR-1 positive bands weredetected by ECL-Western blot detection kit employing rat polyclonalanti-HCCR-1 serum as in Example 14.

The result in FIG. 28 demonstrates that HCCR-1 protein having a relativemolecular mass of approximately 40,000 (M_(r) ˜40K) begins to beoverexpressed at fetal 18-day remains at a high expression level up topostnatal 14-day, and decreases to a very low level in adult rat kidney.In FIG. 28, F and P denote fetal and postnatal, respectively.

A 20-day-old fetal rat kidney was subjected to an immunohistochemicalstaining as in Example 15. As revealed in FIG. 29 which shows a stainedsection of the rat kidney (Magnification, ×42), HCCR-1 antibody stainedthe collecting ducts only (medulla on the left side), which are derivedfrom the ureteric bud (Saxen, L. Organogenesis of the kidney. 88-128(Cambridge University Press, Cambridge, United Kingdom, 1987); Coles, H.S., et al., Development 118, 777-784 (1993)). The developing nephrons inthe cortex were not stained (nephrogenic zone on the right side).

Further, a 18-day-old fetal rat kidney was subjected to animmunohistochemical staining as in Example 15 and, then, observed undera differential-interference contrast microscope. As shown in FIG. 30,the basolateral plasma membranes of medullary collecting duct wereespecially reactive to HCCR-1 antibody (Magnification, ×220).

Because nephrogenesis is stimulated by a distinct ureteric signal,diffusion-limited basolateral molecules (Barasch, J., et al., Am. J.Physiol. 271, F50-F61 (1996)), which trigger mesenchymal to epithelialconversion, it is presumed that the HCCR-1 product may be amesenchyme-derived regulatory factor (Barasch, J. et al., Cell 99,377-386 (1999): Barasch, J. et al., J. Clin. Invest. 103, 1299-1307(1999)) that stimulates morphogenesis of epithelia in the kidneydevelopmental process and mediates interactions between mesenchyme andepithelia during neoplastic transformation.

The present specification includes the appended Sequencing Listing of 47nucleic acid or amino acid sequences. Articles of the patent andscientific periodical literature cited herein are thereby incorporatedin their entity by such citation.

While the embodiments of the subject invention have been described andillustrated, it is obvious that various changes and modifications can bemade therein without departing from the spirit of the present inventionwhich should be limited only by the scope of the appended claims.

1. A human cervical cancer 1 protooncogene having the base sequence ofSEQ ID NO:1 or a fragment thereof.
 2. The fragment of the protooncogeneof claim 1 having a base sequence corresponding to base Nos. 9 to 1088of SEQ ID NO:1.
 3. A protein having the amino acid sequence of SEQ ID:2or a fragment thereof.
 4. A vector comprising the protooncogene orfragment of claim
 1. 5. A microorganism transformed with the vector ofclaim
 4. 6. The microorganism of claim 5, which is E. coli JM109/HCCR-1(Accession No.: KCTC 0667BP).
 7. A process for preparing the protein orfragment of claim 3 comprising culturing the microorganism of claim 5 or6.
 8. A kit for diagnosis of cancer which comprises the protooncogene orfragment of claim 1 or
 2. 9. A kit for diagnosis of cancer whichcomprises the protein or fragment of claim
 3. 10. An anti-sense genehaving a base sequence which is complementary to the sequence of thefull or partial mRNA transcribed from the protooncogene or fragment ofclaim 1 or 2 and being capable of binding the mRNA to inhibit theexpression of said protooncogene or fragment.
 11. The anti-sense gene ofclaim 10 having the base sequence of SEQ ID NO:3.
 12. A process fortreating or preventing cancer in human which comprises administering atherapeutically effective amount of the anti-sense gene of claim 10 or11 to the human.